Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici

• Published in: Brazilian journal of microbiology 2014-01, Vol.45 (1), p.351-358
• Main Authors: Feng, Bao Zhen, Li, Peiqian
• Format: Article
• Language: English
• Subjects: Cloning; Cloning, Molecular; Conserved Sequence; Enzyme Stability; Enzymes; Fungi; Gene Expression; Genomics; Hydrogen-Ion Concentration; Laccase - chemistry; Laccase - genetics; Laccase - isolation & purification; Laccase - metabolism; MICROBIOLOGY; Molecular Weight; Open Reading Frames; Phytophthora - enzymology; Phytophthora - genetics; Pichia - genetics; Protein Sorting Signals - genetics; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Research Paper; Sequence Homology, Amino Acid; Temperature
• ISSN: 1517-8382; 1678-4405; 1678-4405
• DOI: 10.1590/S1517-83822014005000021

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 °C, respectively. The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.